ABSTRACT Non-small cell lung cancer accounts for the most cancer-related deaths worldwide and there is currently a severe lack of treatment options for patients with advanced-stage disease. Patients with lung adenocarcinoma, the most common histological subtype, often have activating mutations in the Epidermal Growth Factor Receptor (EGFR) and can be treated with tyrosine kinase inhibitors (TKIs); however, this treatment is not curative. A new treatment paradigm is emerging with the recent FDA approval of osimertinib, a mutant-specific third generation EGFR TKI, as a first-line therapy for advanced EGFR mutant non-small cell lung cancer patients. Recent data from an ongoing clinical trial shows an impressive 19-month progression- free survival in these patients, compared with 10 months in patients treated with a first generation EGFR TKI. There are currently no approved therapeutic strategies to overcome resistance to osimertinib, highlighting the need for novel strategies to improve patient survival. However, very little is known about the mechanisms of acquired resistance to first-line osimertinib and this must be explored before an optimal second-line treatment strategy can be developed. We hypothesize that resistance to first-line osimertinib is mediated primarily through secondary mutations in EGFR, and tumors with these mutations exhibit different patterns of sensitivity to first or second generation EGFR TKIs. Our hypothesis is based on: 1) published data from cell lines showing that the secondary mutations in EGFR are differentially sensitive to first and second generation EGFR TKIs and 2) preliminary work from our lab indicating that osimertinib-resistant tumors differentially respond to other TKIs in vivo based on the specific mutation present. To achieve our goal, we propose to further uncover the mechanisms of acquired resistance to first-line osimertinib and the biochemical or signaling basis of this resistance. Additionally, we will establish the sensitivity of these secondary EGFR mutations to other EGFR TKIs and the method through which they confer their resistance.